Flow Cytometry

Question 1

Forward scatter

Answer 1

measures cell size

Less in dead cells

Question 2

Side scatter

Answer 2

Measures relative granularity

More in dead cells

Question 3

Single-colour fluorescent staining

Answer 3

Plotted as histogram 
# cells vs log intensity of fluorescence

Question 4

Two-colour fluorescent staining

Answer 4

Plotted as a dot-plot or contour plot

Log of intensity vs log of intensity

Question 5

PE-conjugated anti-TCR

Answer 5

bright yellow

Question 6

FITC-conjugated anti-CD4

Answer 6

green

Question 7

PE-Cy5-conjugated anti-CD8

Answer 7

dark red

Question 8

Flow cytometer

Answer 8

Stained cells flow through center of a narrow stream of buffer or medium that emerges from vibrating nozzle in droplets of no more than 1 cell/drop
A laser beam is focused on the stream so that it strikes one cell at a time
Can have up to 10 lasers=10diff fluorochromes
Scattered light and fluorescent light detected by photomultiplier tubes –> transmitted to computer

Question 9

sodium azide

Answer 9

Added to staining buffer
prevents capping and endocytosing of surface molecules aggregated by Ab during staining
Cells are also kept at 4C or on ice

Question 10

Control Ab

Answer 10

Conjugated with PE
Conj w FITC
Conj w PE-Cy5
Used with WT/KO spleen and thymus

Question 11

Compensation

Answer 11

Overlap of emission spectra
can compensate by only looking at single stained cells
Color Compensation is defined as the subtraction of a percentage of the signal from one fluorescent light sensor detector from the signal of another fluorescence light sensor to correct overlap of one dye’s emission measurement.

Question 12

B2 microglobulin -/- mouse

Answer 12

Doesn’t express Class 1 MHC –> No CD8+

Question 13

Intracellular staining

Answer 13

Ags are usually signaling molecules (e.g. NF-κB) or proteins that will be secreted (e.g. cytokines)
Permeabilization –> low concentrations of mild detergents such as Saponin or Tween (detergents allow antibodies to cross membranes without lysing cells access the cytoplasm and potentially inside the ER and Golgi)

Question 14

Analytical measurements for DNA quantitation

Answer 14

Cells are fixed so that dyes can easily enter cells. Relative DNA quantitation is given based on the amount of dye staining.
The DNA quantity can be used to determine stage in cell cycle (G0/G1, S, G2) or the induction of apoptosis
Common dyes = Propidium iodide (PI) and acridine orange

Question 15

Gating

Answer 15

term for drawing a computer generated parameter (limiting your analysis) around cells with specific size/granularity or staining characteristics
Almost always gate on the FSC/SSC to select uniform cell size and granularity for analysis of fluorescence
Often based on past experience but when first analyzing a new population of cells it can be important to determine what cell types are located where on your FSC/SSC dot plot
May also gate on positive or negative fluorescent staining cells as way to select certain cell types in your analysis.

Question 16

Cell Sorting

Answer 16

Separate cells based on surface expression for further experiments.
Example: Spleen cells from mouse are stained with anti-CD3 antibody conjugated with green FITC. Cells are run through sorting flow cytometer. CD3 positive staining cells have charge added to their surface, moved by magnet and collected in a separate tube. CD3+ sorted cells can be put back into cell culture or put into other mice (adoptive transfer).